The OVERALL OBJECTIVE of the proposed investigation is to elucidate the role of the interferon-inducible double-stranded RNA-dependent protein kinase (PKR) in the actions which natural and recombinant interferons mediate on viral and host functions. The SPECIFIC AIMS of our proposed continuation investigation of protein phosphorylation and interferon (IFN) action are as follows: (1). To further delineate the structure of the 5'-flanking region of the Pkr gene required for interferon-inducible as well as basal transcriptional activity. To map the major Pkr transcription sites, using RNA from tissues of uninfected and infected mice and from untreated and IFN-treated cells. To elucidate the importance of potential protein binding sites in Pkr promoter activity, with emphasis placed on proteins binding the novel 15 nucleotide DNA element designated KCS which so far is unique to the human and mouse Pkr promoters. To examine the ability ofcytokines and factors other than IFNs to modulate P_ expression. (2). To further characterize the biochemical and biophysical properties of the PKR kinase. To attempt to identif3' definitively the sites of PKR autophosphorylation associated with activation. To characterize the structural basis and functional significance of PKR protein interactions, and to continue efforts to obtain a crystal structure of PKR with the hope that the structure will provide further insight into function. (3). To characterize the expression and function ofwildtype and mutant PKR proteins in cell culture. To assess the effect of singular expression of PKR forms in transfected PKR-deficient cells. To use RNase protection and DNA microarray approaches to gain insights into role of PKR on transcript profiles in untreated, IFN-treated, and infected cells from two strains of mice. The health relatedness of the proposed research stems from the likelihood that the work may contribute to a better understanding of regulatory mechanisms operative in normal cells as well as virus-infected cells. Elucidation of the actions of IFN at the molecular level is of immediate importance in view of the use of IFN in the clinic.